Process for producing 7-(5-amino-5-carboxyvaleramido)-7-methoxycephalosporamic acid

ABSTRACT

ANTIBIOTIC AL6884 AND ITS SALTS, HAVING ANTIBACTERIAL AND ANTHELMINTIC ACTIVITY, PREPARED BY FERMENTATION OF STREPTOMYCES LIPMANII NRRL 35984.

March 6, 1973 R. l.. HAMILL ETAL 3,719,563

PROCESS I-OH PRODUCING 7(5'AMINOS CARBOXYVALERMIDO)7-METHOXYCEPHALOSPORANIC ACID Filed Aug. s. 1970 NOISSIWSNVUl LNBSHI-ldUnited States Patent O PROCESS FOR PRODUCING 7 (S-AMINO-S-CAR-BOXYVALERAMIDO) 7 METHOXYCEPHALO- SPORAMIC ACID Robert L. Hamill, NewRoss, and Calvin E. Higgeus and Marvin M. Hoehn, Indianapolis, Ind.,assiguors to Eli Lillyfand Company, Indianapolis, Ind.Continuation-impart of application Ser. No. 847,923,

Aug. 6, 1969. This application Aug. 3, 1970, Ser.

Int. Cl. C12d 9/00 U.S. Cl. 195-80 R 3 Claims ABSTRACT F THE DISCLOSUREAntibiotic A16884 and its salts, having antibacterial and anthelminticactivity, prepared by fermentation of Streptomyces lz'pmam'i NRRL 35984.

CROSS-REFERENCE TO RELATED APPLICATION This is a continuation-impart ofour copending application, Ser. No. 847,923, filed Aug. 6, 1969 andabandoned after the tiling of this application.

SUMMARY OF THE INVENTION Antibiotic A16884 is a new antibiotic producedby the fermentation of an antibiotic A16884-producing strain ofStreptomyces lipmanii. The salts of A16884 are readily obtained byreaction of A16884 with a suitable acid or base. Antibiotic A168 84 andits salts exhibits antibacterial and anthelmintic activity. Theantibacterial activity is exhibited against both gram-negative andgram-positive organisms, as well as against plant-pathogenic organisms.

DESCRIPTION OF PREFERRED EMBODIMENTS Antibiotic A16884, asulfur-containing peptide antibiotic, is an amphoteric molecule producedby cultivating under controlled conditions a hitherto undescribed strainof Streptomyces lz'pmanz'i NRRL 3584.

As is the case with many antibiotic-producing cultures, fermentation ofan antibiotic A16884-producing strain of Streptomyces lipmam results inthe production of a number of antibiotic substances. Antibiotic A1684 isone of these substances. Other substances are either relatively unstableor are present in only very minor quantities.

Antibiotic A16884 can be utilized as such or as a salt, for example, anacid addition salt or a salt with a cation. In the instance of a saltwith a cation, the salt can be either a mono or di salt. It is oftenpreferred to prepare salts directly in the purification process so thatthe antibiotic as separated is in salt form. Antibiotic A16884 has beenseparated in this manner, and for that reason, is hereinbelowcharacterized as the mouoammonium salt.

The mouoammonium salt of antibiotic A16884 is a white, amorphous solid,decomposing at about 180 C., very soluble in water, soluble indimethylsulfoxide (DMSO), slightly soluble in lower alkanols, andessentially insoluble in acetonitrile and other organic solvents. Thespecific optical rotation [a]D25 of the monoammonium salt of antibioticA16884, dried at room tern- ICC perature in vacuo over anhydrous calciumchloride for about 15 hours, was found to be-f-140.9 (C=1 percent, w./v.in water).

Electrometic titration of the mouoammonium salt of antibiotic A16884 ina 66 percent dimethyl formamidewater solution at an initial pH of 6.6revealed the presence of four titratable groups: pKa1=3.5; pKa2=5.2; andpKa3-9.2; and pKa4=10.3. On like titration of a later sample, except atan initial pH of 5.8, the respective values were pKal=3.9; pKa2=5.3;pKa3=9.2; and pK4=10.5. When the mouoammonium salt of `antibiotic A16884is converted to the acid form, the pK'a at 9.2 disappears. The molecularweight of the mouoammonium salt calculated from the titration data isabout 435.

Elemental analysis of the mouoammonium salt of A16884, dried in vacuo atabout '80 C. over phosphorus peutoxide, gave the following values:

Element: Percent Carbon 44.01 Hydrogen 5.73 Nitrogen 10.65 Oxygen 31.27Sulfur 6.86

Analysis shows a methoxyl content of 6.64 percent, and an acetyl contentof 9.23 percent; and a Van Slyke test for amino nitrogen shows 5.09percent.

The infrared absorption spectrum of the monoammonium salt of antibioticA16884 in a mineral oil mull is shown in the figure of the accompanyingdrawing. The distinguishable bands in the infrared spectrum over therange of 2.0 to 15.0 microns are as follows: 3.18 (broad band), 5.66,6.26, 6.57, 6.89, 7.15, 7.28, 7.40, 7.73, 8.00,

8.14, 8.79, 9.24, 9.65, 9.79, and 10.4 microns.

The ultraviolet absorption spectrum of the monoammonium salt ofantibiotic A16884 in aqueous solution shows absorption maxima at 242circular dichroism was also measured in aqueous solution and showed apositive Cotton effect at 263 ma and a negative Cotton effect at 236mit.

Paper chromatography of the mouoammonium salt of antibiotic A16884 onWhatman No. l paper gave an Rf value of 0.79 in a solvent system ofpropanol, acetonitrile, and water in a volume ratio of 1: 1:1.Bioautographs were obtained by placing the Vpaper chromatograph on agarplates seeded with sensitive organisms, such as Salmonella gallnarum, astest organisms.

The NMR spectrum of A16884 in D20 showed the following characteristics:5.16 p.p.m. (1H, singlet); 4.86, 4.68 p.p.m. (2H, AB quartet, l- -12.5HL); 3.9-3.7 p.p.m. (1H, multiplet); 3.67, 3.29 p.p.m. (2H, AB quartet,1:18 Hz.); 3.53 p.p.m. (3H, singlet); 2.6-2.3 (2H, multiplet); 2.10p.p.m. (3H, singlet); 2.1-1.6 p.p.m. (4H, multiplet).

Paper chromatography of the monoammonium salt was also carried out inother solvent systems with the following results:

Solvent system: Rf value Ethanol:water (80:20) with 1.5% sodiumchloride, paper impregnated with l N sodium sulfate .58Methanolzpropanokwater (6:2:1), paper buffered with 0.75 M potassiumphosphate,

pH 4.0 .21 Propanol pyridine acetic acid: acetonitrile water(45:30:9:40:36) .40 Tert-amyl alcohol:acetone:water (251:2) .40 Ethylacetate2acetic acidrwater (311:1) .36

Methyl ethyl ketonezwater (92.8), paper buffered with 0.1 N sodiumacetate, pH 4.6 Immobile Propanolzwater (70:30) .30 Butanol saturatedwith water Immobile Butanol saturated with water plus 2%p-toluenesulfonic acid .60

When the monoammonium salt of A16884 is subjected to thin-layerchromatography on silica gel plates in 70 percent aqueous acetonitrile,utilizing a ninhydrin spray as a detector, it has an Rf value of 0.47;on cellulose plates in 70 percent aqueous acetonitrile, utilizing thesame procedure for detection, it has an Rf value of 0.45.

Amino acid analysis of an acid hydrolysate of antibiotic A16884, run bythe Spackman-Moore-Stein technique, showed two ninhydrin reacting peaks,one of which was eluted identically with glycine (0.758 umoles/mg), theother of which was eluted just prior to glycine and was identified asa-aminoadipic acid (2.39 arnoles/mg) On like analysis of a later sample,the values observed were 0.49 ,amoles/mg. and 1.2 moles/mg.,respectively.

A number of qualitative chemical tests have been carried out with theantibiotic A16884. Antibiotic A16884 gives a positive test withninhydrin, Pan Dutscher, Benedict, Molisch, iodine and dansyl chloridereagents, but not with Fehling, ferrie chloride, biuret, and Sakaguchireagents.

The monoammonium salt of antibiotic Al6884 is stable at pH 3-9 at 5YC.for 8 days; relatively stable at pH 3-9 at 25 IC. for 4 days; andunstable at varying pH values at 100 C. Within 5 minutes. Biologicalactivity is slowly lost at pH 3-9 at a temperature of 37 C., half beinglost at 4 days.

Based on the various foregoing physical characteristics the structure ofantibiotic A16884 has been determined to be as follows:

COOH which structure is designated7-(5-amino-5-carboxyvaleramido)-7-methoxycephalosporanic acid.

Antibiotic A16884 has an inhibitory action against the growth of bothgram-positive and gram-negative bacteria. The levels at which partiallypuried monoammonium salt of antibiotic Al6884 shows inhibition againstthe growth of illustrative organisms are set forth in Table I. Theinhibitory levels were determined by the agar-dilution test or by thebroth-dilution test (identified in the table by the letters ad and 1nd,respectively).

In the agar-dilution test, the test organism was streaked on a series ofagar plates containing various concentrations of the monoammonium saltof antibiotic A16884 to determine -the minimum concentration in mcg/ml.(micrograms per milliliter) in the agar substrate which inhibited thegrowth of the organism over a period of forty-eight hours (seventy-twohours in the case of the plant pathogen organisms).

In the broth-dilution test, a series of tubes containing nutrient brothcontaining varied concentrations of the ammonium salt of antibioticA16884 were inoculated with the test organism to determine the minimumconcentration of the monoammonium salt of A16884 in mcg./ rnl. in thebroth substrate which inhibited organism growth for a period of abouttwenty hours.

TABLE I Inhibitory concentration Test organism: mcg/m1. Escherichia coliEC 0127 ad `6.25 Proteus PR6 ad 1.5 6 Proteus PR4 ad 3.12 Salmonellatyphimurium 54 ad 3.12 Salmonella typhosa T63 ad 1.56 Staphylococcusaureus 3055 ad 50.00 Staphylococcus aureus 3150 ad 50.00 Pseudomonasaeruginosa X239 ad 50.00 Salmonella /iexneri SH3 ad 6.25 Klebsiellaaerobacter Kl ad 6.25 Klebsiella aerobacter KA14 ad 1.56 Mycobacteriumavium X ad 50.00 Streptococcus pyogenes C203 ad 3.12 Bacillus subtilisXl2.1 ad 3.12 Neurospora sp. M45-846 ad 50.00 Sarcina lutea X186 ad 6.25Escherichia coli ECO127 bd 7.80 Klebsiella aerobacter KA14 ..bd 15.60Salmonella typhosa SA12 bd-- 31.20

No binding by horse serum was noted in any of the above tests.

As can be seen from the above table, antibiotic A16884 as themonoammonium salt exhibits activity against gram-positive andgram-negative bacterial organisms.

More highly puried monoammonium salt of antibiotic A1'6884 was furtherevaluated for antibacterial activity in a test employing the brothdilution technique described above. The results, expressed in terms ofthe minimum number of micrograms per milliliter required to obtaininhibition, were as set forth below in Table II.

1 ND =Not done.

Antibiotic A16884 and its salts also exhibit in vivo activity against anumber of the above organisms and hence are useful in controllinginfections caused by such organisms in host animals. Partially purifiedantibiotic A16884 as the monoammonium salt exhibited an EDu of 23mg./kg. in mice infected with Proteus PR6, and an EDS@ of 33.8 mg./kg.in mice infected with Shigella SH3; more highly purified Al6884 as themonoammonium salt exhibited an ED50 of 3.64 mg./kg. in mice infectedwith Escherichia coli EC14, an ED50 of 23 Ing/kg. in mice infected withSalmonella typhosa SA12, and an ED50 of 93.4 mg./kg. in mice infectedwith Klebsiella pneumoniae K1. Administration was by the sub-cutaneousroute.

As noted hereinabove, antibiotic A16884 and its salts exhibitanthelmintic activity in addition to antibacterial actvity. Henceantibiotic A16884 or the salt thereof can be administered towarm-blooded animals to control various internal parasites, particularlystomach and intestinal worms such as Ascars lumbricodes var. suum,Nematospirodes dubius, Aspiculurs tetraptera, Syphacia obvelata, and thelike. The administration is `preferably by the oral route, for example,by inclusion of antibiotic A16884 or `a salt in animal feed, byadministration of tablets, drenches, etc. `containing A16884 or a salt,or by other means. In general, doses of from 1 to 500 milligrams perkilogram or more of animal body weight are effective in single doseadministration. Where antibiotic A16884 or a salt thereof is Asuppliedas a constituent of a regular feed, concentrations of from 0.0001 to0.05 percent or more give good results. A preferred range ofconcentration of antibiotic A16884 or a salt thereof in feeds is from0.01 to 0.05 percent.

The anthelmintic activity of antibiotic A16884 is illustrated by thefollowing evaluations.

lIn a rst evaluation, antibiotic A16884 monoammoni- `urn salt wasadministered in a single dose by gavage to each of two mice infectedwith Aspculurs tetraptera and ySyphaca obvelata (pinworms). The dose was500 milligrams of antibiotic A16884 monoammonium salt per kilogram ofindividual animal body weight, administered in a suspension ofphysiological saline containing 0.125 per- -cent of methylcellulose assuspending agent. A control group of mice infected with Aspiculuristetraptera and Synphaca obvelata was employed in the evaluation. Bothgroups were maintained under normal laboratory condi- Ations forforty-eight hours, following the dosing of the treated group. All micewere then sacried and examined .to determine the presence and numbers ofpinworrns, `which were as reported in the following table:

500 Ing/kg 2. 5

In another evalution, antibiotic A16884 monoammonium salt was mixed withstandard mouse feed to obtain a -plurality of treated feeds, containingantibiotic A16884 monoamrnonium salt in concentrations of 0.005, 0.01,and 0.05 percent by Weight. The feeds were utilized as diets forseparate groups of mice, five mice per group. About twenty-four hoursafter initiation of the feeding, the mice were infected with Ascarz'slumbrz'coides var. suum ova. `Another group of live mice was fed thenon-medicated feed to serve as a control but was similarly infected atthe Same time with Ascars lumbrcoz'des var. summ. All groups were fedtheir respective feed and maintained under normal laboratory conditionsfor a period of ten days, at which time, all mice were takenoff feed. Onthe eleventh day, all mice were sacrificed and the lungs examined todetermine the presence and, if present, numbers of lesions of Ascarislumbricodes var. suum.

The level of antibiotic A16884 monoammonium salt in the diet and theaverage number of lung lesions per animal in each group are set forth inthe following table:

TABLE IV i Average number of lung `lesions Group per group Control. ..72. 2 Antibiotic A16884 monoammonium salt at 0. 05 0.5 Antibiotic A16884monoammonium salt at 0.

Antlobiotic A16884 monoammonium salt O.

Antibiotic A16884 can be produced by culturing a 4newly found andhitherto undescribed organism strain to agar slants. The agar slantswere then incubated to provide suitable amounts of inoculum for theproduction of antibiotic A16884.

The actinomycete used according to this invention for the production ofantibiotic A16884 has been designated as a strain of Streptomyces lipmanWaksman and (hirtis.

The novel organism capable of producing antibiotic A16884 has beenplaced on permanent deposit without restriction as to availability withthe culture collection of the Northern Utilization Research andDevelopment Division, Agricultural Research Service, U.S. Department ofAgriculture (Formerly Northern Regional Research Laboratories), Peoria,Ill. 61604, and is available to the pubnp under cinture No. N-RRL 3584.

The characteristics of Streptomyces lpmanz'z' NRRL 3584 are given in thefollowing tables. The methods recommended for the InternationalStreptomyces Project (Shirling et al., Methods for Characterization ofStreptomyces Species, Intern. Bull. Systematic Bacteriol. 16: 313-340[1966]) for thecharacterization of Streptomyces species have been usedalong with certain supplementary tests. Color names were assignedaccording to the ISCC- NBS method described by Kelly et al. in TheISCC-NBS Method of Designating Colors and a Dictionary of Color Names(U.S. Department oi" Commerce Circ. 4553, Washington, D.C. l1955).Figures in parenthesis refer to the Tresner and Backus color series(Tresner et al., System of Color Wheels for Streptomyces Taxonomy, Appl.Microbiol. l1: 335-338 [1963]) and color tab designations areunderlined. The vMaerz and Paul color blocks (Maerz -et al., Dictionaryof Color (McGraw-Hill Book Co., Inc., New York, 1950) are enclosed inbrackets. Cultures were -grown at 30 C. for 14 days unless notedotherwise.

to ilexuous with occasional hooks produced; spores are short,cylindrical, 0.5-1.5u x 1.0-2.5;4, and occur usually in chains o 3-10and occasionally from 10-50. Sporcs are smooth in outline as observed byelectron microscopy.

Culture characteristics on:

ISP No. 2` (Yeast-malt extract Growth moderate, reverse dark grayishbrown [8H9]; aerial mycelium pale yellow (Y) 2db.

ISP No. 3 (Oatmeal agar) Growth moderate, reverse dark grayish yellow[13E4]; aerial mycelium moderate, white (W) 13ba to pale yellow (Y) 2db.

ISP No. 4 (Inorganic salts and Growth moderate, reverse brownsolublestarch agar). ish gray [7C7]; aerial myeelium moderate, pale yellow (Y)2db.

ISP No. 5 (Glycerol-asparagine Growth abundant, reverse light agar).yellowish brown [13I7]; aerial mycelium abundant, grayish yellowish pink(R) 5de.

Tornato paste-oatmeal agar Growth abundant, reverse grayish yellowishbrown [15E8]; aerial mycelium abundant, yellowish gray (GY) Zdc.

Emersons agar Growth moderate, reverse dark grayish yellowish brown[8E9]; aerial myeelium and spores absent.

Bennetts agar Growthabundant,revcrse medium yellowish brown [14137];aerial mycelium abundant, grayish yellow (R) 3ec.

agar) Czapeks agar Growth scant, white; scant aerial mycelium (W) 13ba.Glucose-asparagine agar Growth abundant, reverse grayish TABLEV--Continued Property observed Characteristics of A16884 Physiology:

Action on milk Coagulation, peptonization. Nitrate reduction. Positive.Melanin production:

Peptone-iron agar Negative. Tryptone-yeast ext. broth D Abundant growthand sporulation at 26 C. and 30 C.; slight ggpvth at 37 C.; no growth atTemperature requirements on tomato paste-oatmeal agar.

Response of vegetative color to pH change:

0.05 N BICI Brovnish-gray pigment changes to re 0.05 N NaOH No change.Gelatin liquefaction 100%.

In Table IV are set forth the results of carbon utilization testscarried out on organism NRRL 3584. In the table, the following symbolsare employed: -{-1=growth and utilization, and -=no growth, noutilization.

TABLE VI Carbon utilization pattern for NRRL 3584 As noted above,antibiotic Al6884 can be produced by the cultivation of NRRL 3584. Theculture medium employed in producing antibiotic Al6884 by cultivation ofthe above-identified organism can be any one of several media, since, asis apparent from the above-described utilization tests, the organism iscapable of utilizing different energy sources, However, for economy ofproduction, maximum yield of antibiotic, and ease of isolation of theantibiotic, certain relatively simple nutrient sources are preferable.For example, the media which are useful in the production of theantibiotic include an assimilable source of carbon such as glucose,starch, glycerine, molasses, dextrin, and the like. The preferred sourceof carbon is glucose. Additionally, employable media include a source ofassimilable nitrogen such as soybean meal, corn steep solids, yeast,cottonseed meal, beef extract, peptones, (meat or soy) casein, aminoacid mixtures, and the like. Preferred sources of nitrogen are peptones,soybean meal, amino acid mixtures, and the like. Among the nutrientinorganic salts which can be incorporated in the culture media are thecustomary salts capable of yielding sodium, potassium, ammonium,calcium, phosphate, sulfate, chloride, carbonate, and like ions.

Minor elements necessary for optimum growth and development of theorganism used for the production of antibiotic Al6884 can also beincluded in the culture medium. Such trace elements commonly occur asimpurities in the other constituents of the medium in amounts sumcent tomeet the growth requirements of the actinomycete employed in thisinvention.

The initial pH of the culture medium can be varied. However, it has beenfound desirable that the initial pH of the medium be between 6.5 and7.2. As has been observed with other actinomycetes, the pH of the mediumgradually increases throughout the growth period of the organism whilethe antibiotic is being produced, and may attain a level of from 6.7 to7.5 or above, the iinal pH being dependent at least in part on theinitial pH of the medium, the buffers present in the medium, and theperiod of time the organism is permitted to grow.

submerged, aerobic cultural conditions are the conditions of choice forthe production of antibiotic Al6884. For preparation of relatively smallamounts, shake flask and surface culture in bottles can be employed; butfor the preparation of large amounts, submerged aerobic culture insterile tanks is preferred. The medium in the sterile tank can beinoculated with a sporulated suspension; but because of the growing lagexperienced when a sporulated suspension is used as the inoculum, thevegetative form of the culture is preferred. By thus avoiding the growthlag, more eiiicient use of the fermentation equipment is realized.Accordingly, it is desirable first to produce a vegetative inoculum ofthe organism by inoculating a relatively small quantity of culturemedium with the spore form of the organism; and when a young, activevegetative inoculum has been obtained, to transfer the vegetativeinoculum aseptically to the large tank. The medium in which thevegetative inoculum is produced can be either the same as or differentfrom the medium utilized for the large-scale production of antibioticA16884.

The organism which produces antibiotic Al6884 will grow over a widetemperature range between 25-37 C. Optimal production of A16884 seems tooccur at temperatures of 26-30 C. In general, maximum production of theantibiotic occurs within about 36-72 hours after inoculation of theculture medium.

As is customary in aerobic, submerged culture processes, sterile air isblown through the culture medium. For eicient growth of the organism andantibiotic A16884 production, the volume of air employed in the tankproduction of A16884 is from 0.2 to 0.4 volume of air per minute pervolume of culture. The preferred volume is 0.40 volume of air per minuteper Volume of culture medium.

The concentration of antibiotic activity in the culture medium can befollowed readily during the fermentation period by testing samples ofthe culture medium for their inhibitory activity against the growth oforganisms known to be inhibited by the presence of antibiotic A16884.The organisms Saz'cina lutea and Salmonella gazllz'narumy have beenfound to be useful for this purpose. The testing of the samples can becarried out by the well-known turbidometric or diso-plate methods.

In general, maximum production of Al6884 occurs within one to three daysafter inoculation of the culture medium in submerged aerobic culture orshake flask culture processes.

The antibiotic activity produced during the fermentation of A16884occurs in the antibiotic broth. Accordingly, isolation techniquesemployed in the production of Al6884 are designed to permit maximumrecovery of the antibiotic from the broth. Thus, for example, myceliumand undissolved solids are removed from the fermentation broth byconventional means such as ltration or centrifugation, and antibioticAl6884 can be recovered from the filtered or centrifuged broth byemploying extraction or adsorption technique.

For the recovery of A16884 by adsorption techniques, various adsorbentsand ion exchange resins can be used', for example, carbon, silica gel,alumina, and ion exchange resins. Antibiotic Al6884 as obtained fromfermentation may be either amphoteric or salt form, depending uponfermentation conditions. Regardless of which form, it can be adsorbedonto one of the above or similar adsorbents from solution in a suitablesolvent. The adsorbed antibiotic A16884 or salt can be eluted from theadsorbent by suitable elution techniques, such as by washing theadsorbent on which the antibiotic A16S84 or salt thereof is adsorbedwith a solvent. Where the elution is carried out by washing with asolution of, e.g., ammonium formate or sodium acetate, the processresults in elution of antibiotic A16884 as the ammonium or sodium salt,respectively. Such salts are readily converted back to antibiotic A16884in conventional procedures. In the foregoing recovery procedure,microcrystalline cellulose can also be used as adsorbent.

Salts of antibiotic A16884 other than ammonium or alkali metal arepreferably prepared by conventional reaction of antibiotic A16884 inunmodied amphoteric form with the respective acid or base. Thus, inpreparing acid addition salts, antibiotic A16884 is reacted with aninorganic or organic acid. Representative suitable acids includehydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid,phosphoric acid, acetic acid, benzoic acid, sulfamic acid, tartaricacid, citric acid, maleic acid, succinic acid, ascorbic acid, andglycolic acid.

Antibiotic A16884 also forms salts with cations by reaction of A16884 inunmodified, amphoteric form with inorganic and organic bases and salts.Exemplary of these salts are ammonium and substituted ammonium salts;alkali metal salts, such as sodium, potassium, lithium, cesium andrubidium; alkaline earth metal salts such as calcium, strontium, andbarium; and salts with other metals such as aluminum, copper, zinc,magnesium, and silver. In respect to organic bases, the identity of thebase is not critical, although in general, a base having a pH of,numerically, 3.0 or above in water is preferred. Representative suitableorganic bases include benzylamine, methylamine, diethylaminc,triethylamine, procaine, dilsopropylamine, ethanolamine,cyclohexylamine, dicyclohexylamine, diphenylamine, di-n-butylamine,quinoline, and pyridylamine.

The salts of antibiotic A164884 which are pharmaceutically acceptableare generally preferred. However, all salts are useful as intermediatesin the production, separation, and purification of antibiotic Al6884.For therapeutic purposes, either cationic or anionic pharmaceuticallyacceptable salts are generally equivalent to antibiotic A16884; however,particular salts are occasionally preferred due to a favorable property,such as solubility, conferred by the salt-forming moiety.

In order to illustrate more fully the operation of the invention, thefollowing examples are provided by way of illustration.

EXAMPLE 1 Shake tiask production of antibiotic A168234 A sporulatedculture of Streptomyces lipmam'i NRRL 3584 was produced by growing theorganism on a nutrient agar slant having the following composition:

Grams Dextrin 10.00 Yeast extract 1.00

Hydrolyzed casein (N-Z Amine-Type A, Sheield lChemical Company) 2.00Beef extract 1.00 Meer agar (washed three times) 20.00 Deionized water,1 liter.

The pH of the medium was adjusted to pH 7.0 by the addition of sodiumhydroxide.

The agar slant was inoculated with spores of Streptomyces lipmani NRRL3584 and was incubated for 6 days at 30 C. The agar slant was thencovered with sterile distilled water and gently scraped to remove thespores and cells as an aqueous suspension thereof. One milliliter of theresulting suspension was used to inoculate each 100 ml. portion of avegetative medium having the following composition:

Grams Glucose 15.00 Soybean meal 15.00 Cornsteep solids 5.00 Calciumcarbonate 2.00 Sodium chloride 5.00

Deionized water, 1 liter.

The pH of the vegetative medium was adjusted to pH 6.7 by the additionof sodium hydroxide.

The vegetative inoculum was shaken for 36 hours at 30 C. on a reciprocalshaker with a two-inch stroke at 108 r.p.m. The inoculum so prepared wasthen utilized in the production of A16884 as follows.

10 A production medium was prepared having the following composition:

Grams Soybean meal 15.00 Casein 1.00 Sodium nitrate 3.00 Glucose syrup(50 percent glucose) 20.00

Tap water, 1 liter.

EXAMPLE 2 Antibiotic A16884 was produced according to Vthe process ofExample l, but utilizing a production medium having the followingcomposition:

Grams Distillers solubles (Nadrisol) 5,00 Soybean Iflour (Nutrisoy 200D)5.00 Peanut meal 5.00 Blackstrap molasses 5.00 Oatmeal 5.00 Glycerol10.00

Tap water, 1 liter.

and utilizing instead of a rotary shaker a reciprocal shaker operatingat 108 strokes per minute.

EXAMPLE 3 Antibiotic -A16884 was produced according to the process ofExample 1, but utilizing a production medium having the followingcomposition:

Grams yOatmeal 20.00 Glycerol 10.00

Tap Water, 1 liter.

EXAMPLE 4 Antibiotic A16884 was produced according to the process ofExample l, but utilizing a production medium having the followingcomposition:

Grams Cottonseed our 20.00 Glycerol 10.00 Glucose 5.00

Antibiotic A16884 was produced according to the process of Example 1 bututilizing a production medium having the following composition:

Grams Glucose 20.00 Soluble starch 10.00 Peptone (Wilsons 159) 30.00

Hydrolyzed casein (N-Z amine-type A, Sheffield 'Chemical Co.) 4.00Magnesium sulfate heptahydrate 5.00 Sodium carbonate 2.00

Tap water, 1100 ml.

EXAMPLE 6 Another sporulated culture of Streptomyces lpmanz'i NRRL 3584was produced by growing the organism on a nutrient agar slant. The slantin this instance had the following composition:

Grams Dextrin 10.00 Cottonseed our 10.00 Yeast extract 1.00 Meer agarv25.00

Deionized water, 1000 ml.

The pH of the medium was adjusted, by addition of sodium hydroxide, to7.0.

The agar slant was inoculated with spores of Streptomyces lipman NRRL3584 and incubated for 7 days at 30 C. The agar slants were then scrapedto remove spores to which were added ml. of sterile beef serum. To asterile lyophile tube was then transferred 0.1 ml. of the resultingserum spore suspension; it Was freeze-dried in the form of pellets.

The freeze-dried pellets thus obtained were used to inoculate avegetative medium having the following composition:

Grams Glucose 5.00 Dextrin 10.00 Bacto-tryptone 5.00 Yeast extract 5.00Magnesium sulfate heptahydrate 2.00

Deionized water, 1 liter.

The pH of the medium was 6.7 and was left unadjusted.

EXAMPLE 7 Pilot plant production of antibiotic Al6884 To a 40-literstainless steel fermenter were added 24 liters of a medium having thefollowing composition:

Grams Antifoam A (an antifoaming agent sold by Dow Corning) 0.20 Glucose5.00 Dextrin 700 50.00 Soybean grits 25.00 Molasses, blackstrap 3.00Potassium biphosphate 0.25 Calcium carbonate 2.50

Cold tap water, to liters.

The initial pH was y6.5 and was not adjusted. The medi-um was sterilizedfor minutes at 120 C., cooled, and then inoculated with a five percentvegetative inoculum produced as in Example 6. The fermentation wascarried out at 30 C. for 66 hours, aerated with sterile air at the rateof 0.35 v./v./min., and agitated by a mechanical stirrer operated at420' revolutions per minute. The terminal pH was 7.5.

A165584 was recovered from the broth following the isolation procedureset forth iu Example 8.

EXAMPLE 8 Isolation of crude antibiotic A16884 as the monoammonium saltApproximately 60 liters of broth obtained as reported in Example 7 wasfiltered with the aid of Hyo Super-cel (a diatomaceous earth sold byJohns-Manville Products Corporation). The broth ltrate was passed over a9.6 X 150 cm. column packed with carbon (Pittsburgh Cal. l2 x 40, soldby Pittsburgh Activated Carbon Co). The column was Washed with wateruntil the eluent was colorless, and the activity adsorbed on the carbonWas removed by passing 50 percent aqueous acetone over the column. Thefractions containing the activity were combined, concentrated in vacuoto remove acetone, and applied to a 5.9 X 104 cm. column packed withIRA-68 resin (formate cycle) (an anion exchange resin sold by Rohm andHaas Co. and subsequently washed With formic acid to convert the resinto the formate cycle). The column was Washed With Water until theeilluent was clear and colorless,`and the activity was removed bywashing with 0.1 M ammonium formate solution. The active fractions Werecombined, and passed over a 4.3 x 72 cm. carbon (Pittsburgh 12 x 40)column. The column was washed with six column volumes of Water, and theactivity was eluted with 30 percent aqueous acetonitrile. The activefractions were combined, concentrated in vacuo to remove acetonitrile,and freeze dried. The yield was 25-30 grams of solids.

The freeze-dried preparation was dissolved in a minimum of water andapplied to a 7.2 X 60 cm. column packed with a microcrystallinecellulose product (Avicel, sold by FMC Corporation), suspended in 70percent aqueous acetonitrile, and washed with acetonitrile prior toaddition of the active sample. After application of the sample, thecolumn was Washed with one column volume of acetonitrile, and theactivity was eluted with methanol. The active fractions were combinedand concentrated to approximately 200 milliliters, and the activity wasprecipitated by the addition of 10 volumes of acetone. The precipitatewas filtered, washed withacetone, and dried in vacuo. The yield was 9-12g.

Twenty grams of material obtained as described above was dissolved in aminimum of water and applied to a silica gel column (7.2 x 60 cm.). Thesilica gel (Grade 950 produced by IDavison Chemical) was previouslyWashed with water, then methanol, and suspended in 70 percentacetonitrile for packing the column. After application of the sample,the column was Washed with one column volume of acetonitrile, and theactivity was eluted with 70 percent acetonitrile. The most activefractions were combined, concentrated to dryness in vacuo, and dissolvedin methanol, and the activity was precipitated with l0 volumes ofacetone. The precipitate was filtered, Washed with acetone, and dried invacuo. The yield was 8 g. Less active fractions yielded an additional 6grams.

EXAMPLE 9 Purification of A16884 monoamrnonium salt AOne gram of afreeze-dried preparation prepared as described in Example 8 wasdissolved in four milliliters of Water and applied to a 2 x 60 cm.column packed with milliliters of silica gel Grade 950 in 80 percentaqueous acetonitrile. The column was eluted with acetonitrile: water(4:1). The elution was followed by assay and paper chromatography. As aresult of the elution, a plurality of fractions was obtained. Thefractions containing antibiotic A16884 as the monoarnmonium salt werecombined, concentrated to dryness, dissolved in a small Volume ofdimethylsulfoxide, then in several milliliters of ethanol, and theactivity was precipitated with the addition of excess ether. Theprecipitate was centrifuged and dried in vacuo. The yield of antibioticA16-884 monoammonium salt was 91 mg.

EXAMPLE 10 Preparation of antibiotic A168534 in acid form Two hundredmilligrams of the monoammonium salt of Al6884 were dissolved in 30milliliters of Water, and 6 milliliters of DoWex 501 x 12(H-|) resin(sold by the Dow Chemical Co.) were added. The mixture Was stirred forthirty minutes, filtered, the resin washed With water on the filter, andthe ltrates were combined. The combined filtrate had a pH of 2.7. rlhefiltrate was concentrated in vacuo to about 1 milliliter, 4 millilitersof methanoi were added, and the acid was precipitated by the addition of40 milliliters acetone. The precipitate Was removed by centrifugationand dried in vacuo yielding 35 milligrams of antibiotic A116884 in theacid form. It exhibited pK'as of 3.5, 5.2 and 10.3 when tritrated in 66percent dimethylformamide at an initial pH of 4.5.

EXAMPLE 11 Preparation of disodium salt of A168234 One hundred andeighty milligrams of A16884 monoammom'um salt were dissolved in about 2milliliters Water and the pH was adjusted to 10 with 1 N NaOH. The-solution was concentrated in vacuo to a low volume, 4 millilitersmethanol were added, and the disodium salt was precipitated with theaddition of 40 milliliters acetone. The salt was removed bycentrifugation and dried in vacuo. It exhibited pKas of 3.9, 5.2, and10.5 when titrated in 66 percent dimethylformamide at an initial pH of13 10.4; and when analyzed by atomic absorption analysis, it showed 6percent sodium.

EXAMPLE l2 Preparation of Iantibiotic Al6884 hydrochloride Al6884monoammonium salt (200 mg.) was dissolved in two milliliters of waterand adjusted to pH 2.0 with 1 N HCI. The reaction mixture was thendiluted with 5.0 milliliters of methanol and 50 milliliters of acetoneadded to precipitate the desired antibiotic Al6884 hydrochloride. It wasseparated by centrifugation, washed with acetone, and dried in vacuo.Analysis showed 5.74 percent chlorine and electrometric titration in 66percent dimethylformamide at an initial pH of 5.0 shown titratablegroups at 3.9, 5.2, and 10.4.

EXAMPLE 13 Isolation of crude antibiotic Al6884 as the monosodium saltApproximately 60 liters of broth, obtained as reported in 'Example 7,were ltered with the `aid of Hyo-Supercel. The broth filtrate was passedover a 9.6 x 150 cm. column packed with carbon (Pittsburgh Cal. 12 x40). The column was washed with water until colorless, and the absorbedactivity was removed fby passing 50 percent aqueous acetone over thecolumn. The fractions containing the activity were combined,concentrated in vacuo to remove the acetone, and applied to a 5.9 x 104cm. column packed with IRA-68 (acetate cycle). The column was Washedwith water until the effluent was clear and colorless, and the activitywas removed by washing with 0.1 M sodium acetate. The active fractionswere combined, and passed over a 4.3 x 72 cm. column packed withPittsburgh Cal. (12 x 40) carbon. The column was washed with six columnvolumes of water, and the activity was eluted with 30 percent aqueousacetone. The active fractions were combined, concentrated in vacuo toremove the acetone, and freeze dried. Yield of 20-30` g. Analysis showed2.5 percent sodium.

Antibiotic Al6884, as the monoammonium salt, was evaluated for thecontrol of plant-pathogenic bacterial organisms. In this evaluation,antibiotic A16884 monoammonium salt was formulated in an aqueous sprayformulation at a concentration of 400 parts thereof per million parts byweight of ultimate composition. Thirtyday-old tomato plants were used inthe evaluation, 2 plants/pot. Plants in one pot were treated with thesolution, described above, were allowed to air dry, and were theninoculated with a medium sustaining an active growth of Pseudomonassolanacearum. The plants in the other pot were sprayed with an aqueousspray solution identical with the treating solution described above butlacking the antibiotic, to serve as a control. The plants serving ascontrol were likewise subsequently inoculated. All plants were held for24 hours in a moist chamber, then removed and held for 7 days under goodagricultural conditions. At the end of this seven-day period, all of theplants were observed to determine presence, and if present, degree, ofinfection. The plants treated with antibiotic A16884 monoammonium saltwere completely free of symptoms of disease caused by Pseudomonassolanacearum, Whereas the control plants exhibited extensive symptomsattributable to Pseudomonas solanacearum.

What is claimed is:

1. A method of producing a compound of the formula:

NH2 OCHa U-CHs COOH which method comprises cultivating an organism whichis Streptomyces lipmanii NRRL 3584 in a culture medium containingassimilable sources of carbon, nitrogen, and inorganic salts undersubmerged aerobic conditions until a substantial amount of the saidcompound is produced by said organism in said culture medium.

2. A method according to claim 1 wherein the culture medium ismaintained at a temperature of from approximately 25 C. to approximately37 C. and the growth of the organism is carried out for a period ofapproximately 36 to 72 hours.

3. A method according to claim 2 additionally comprising recovering thesaid compound from said culture medium.

References Cited UNITED STATES PATENTS 3,436,310 4/1969 Arnold et al195-80 X OTHER REFERENCES Miller: The Pfizer Handbook of MicrobialMetabolites, 1961, McGraw-Hill, New York, pp. 610 (1295) and 770.

JOSEPH M. GOLIAN, Primary Examiner U.S. Cl. X.R. 260-243 C

